Cigarette addiction is leading cause of preventable death in the USA. Two major factors contribute to continued smoking and relapse: craving elicited by smoking cues (SCs), and craving elicited by nicotine withdrawal (WD). We demonstrated that SCs activate reward-related circuitry [(ventral striatum, medial orbitofrontal cortex (mOFC)], and linked the brain's response to subsequent smoking behavior. Data were acquired in sated smokers (not in WD) demonstrating that SCs alone can affect responses. Variance in the dopamine transporter SLC6A3 (DAT) gene profoundly affected individual responses: Carriers of a 9 variable number tandem repeat (VNTR) allele exhibited enhanced brain activity in reward-related regions during SC exposure compared to probands homozygous for the 10 VNTR, possibly identifying a SC-vulnerable endophenotype. This may explain some of the heterogeneity in treatment response to smoking cessation medications that act primarily to reduce WD, and further, suggests the existence of pharmaco-responsive endophenotypes. Varenicline, which is twice as effective as other agents, acts on nicotinic cholinergic receptors to modulate the DA system. We present data that varenicline reduces subjective craving and blunts responses to SCs in the 'reward activated' mOFC. Further, effects were inversely correlated with activity of the 'reward evaluating' lateral OFC in the brain at rest. These data imply that the greater efficacy of varenicline may be a function of the reciprocal actions. As such, varenicline is an ideal probe for characterizing the brain and genetic underpinnings of SC vulnerability. Characterization of a SC-vulnerable pharmaco-responsive endophenotype is the overall goal of this application. Towards this goal, we will 1) confirm and extend the finding that 9 VNTR carriers have greater brain and behavioral responses during SC exposure, identifying a SC-vulnerable endophenotype, 2) expand preliminary brain and behavioral findings to characterize a varenicline-responsive endophenotype, and 3) examine the interaction between DAT variance and varenicline- induced brain and behavioral responses to identify a SC-vulnerable pharmaco-responsive endophenotype. Study design: Using an innovative brain/behavioral model that allows for direct comparisons of the effects of pharmacological manipulations over time, providing knowledge of the underlying mechanism of medication effects, data will be acquired prior to and following a 3 week medication regimen. At each time point smokers will be imaged under conditions of rest and during SC exposure. Following imaging sessions, smoking behavior will be monitored using novel naturalistic methods. DNA samples will be analyzed for allelic variance in DA regulating genes. Significance: These studies will definitively link SC-induced limbic activation and smoking behavior; identify brain mechanisms characteristic of effective medication, thus, providing a model to test the predictive validity of novel molecules; and characterize a varenicline-responsive endophenotype in smokers that will potentially lead to personalized treatment strategies and greater smoking cessation success.